Extract of the Family Dioscoreaceae and Composition for Preventing or Treating Peripheral Neuropathy Comprising the Same

ABSTRACT

An extract of the family Dioscoreaceae useful for preventing or treating the peripheral neuropathy; and a pharmaceutical composition or food composition comprising the extract are provided. In addition, a pharmaceutical composition or food composition useful for preventing or treating the peripheral neuropathy comprising a compound isolated from the extract of the family Dioscoreaceae is provided.

TECHNICAL FIELD

The present invention relates to an extract of the family Dioscoreaceaefor preventing or treating the peripheral neuropathy; and apharmaceutical composition or food composition comprising the extract ora compound isolated from the extract.

BACKGROUND ART

Neuropathy is a disease caused by structural or functional abnormalitiesof the nervous system. The nervous system is divided into the centralnervous system which is distributed in the brain and the spinal cord andinvolved in controlling their functions, and the peripheral nervoussystem which is distributed in almost all organs excluding the brain andthe spinal cord and involved in controlling their functions. Theperipheral nervous system is subdivided into the motor nervous system,the sensory nervous system, the autonomic nervous system. A peripheralnerve, where neurites branch out beyond the brain and the spinal cord tothe body, arms, and legs, transmits sensation felt at arms and legs tothe central nerve (brain and spiral cord), and transmits orders of thecentral nerve to muscles.

The peripheral nerve can be injured by various causes, which is calledcollectively as the peripheral neuropathy. The mono-neuropathy refers toa case in which a single peripheral nerve is injured, and the multipleneuropathy refers to a case in which many peripheral nerves are injuredto a similar level. The mono-neuropathy usually occurs when a singleperipheral nerve is abnormally pressed or traumatically injured duringextending to ends of arms and legs. The mono-neuropathy can be treatedwith operations.

The multiple neuropathy can be induced by various causes, such asmetabolic diseases (e.g., diabetes, renal failure, hypothyroidism),drugs (e.g., antitumor agents, antituberculosis drugs) or toxicsubstance intoxication (e.g., Pb, organic solvents), malnutrition (e.g.,vitamin deficiency, alcoholism), connective tissue disorders (e.g.,rheumatoid arthritis, systemic lupus erythematosus), inflammatorydiseases (Guillain-Barre syndrome), or genetically determinedneuropathy. In addition, the multiple neuropathy can be caused bycancers.

Until now, the neuropathy has been treated with drugs used as asymptomatic therapy that improves symptoms only and there are almost nofundamental remedies for the neuropathy. Only epalrestat, an aldosereductase inhibitor, was approved by US Food and Drugs Administration(FDA), with respect to diabetic peripheral neuropathy, one of themultiple neuropathies, but epalrestat is not used due to its lowtherapeutic effects (Foster D W., Harrison's Principles of InternalMedicine 13, p 1979, 1999; Stephen L D, Applied Therapeutics: theclinical use of drugs. 6, p 48.1-48.62, 1996).

Meanwhile, a protein that affects the growth, differentiation, andsurvival of neurons in the central nervous system (CNS) and theperipheral nervous system (PNS) is called collectively as a neurotrophicfactor (NF), which is one of neuron control factors that regulate thegrowth, differentiation, and death of neurons. Examples of the NFinclude a brain-induced neurotrophic factor (BDNF), neurotrophin-3(NT-3), NT-4, and NT-5. These NFs are synthesized at different areas,and have different differentiation, different expression, and differenttarget regions.

A nerve growth factor (NGF), one of the NFs, inhibits the degenerationand death of neurons so as to prevent a decrease in the number ofneurons, to protect neurons from damages, and to retain mature neuronsources (Hefti F., J. Neurosci., 6(8), pp 2155-2162, 1986; andLevi-Montalcini R., et al., Proc. Natl. Acad. Sci. USA, 46, pp 384-391,1960). It is known that when the nervous system normally develops, about50% of neurons on growth are removed by cell death (Raff M C., et al.,Science, 262(5134), pp 695-70, 1993), and that NGFs secreted by a targetcell determine survival of neurons. In order for neurons to survive,grow, and differentiate in a normal state, a growth factor, such as theNGF, is necessarily required. Such feasibility of the NGF has leddevelopment of a recombinant human nerve growth factor in order to treata diabetic neuropathy, one of the multiple neuropathies. However, therecombinant human nerve growth factor is still unsatisfactory in safetyand efficacy (Apfel S C et al., Journal of American Medical Association284(17), pp 2215-2221, 2000).

DISCLOSURE OF INVENTION Technical Problem

The present invention provides herbal extracts and compounds isolatedtherefrom useful for preventing or treating the peripheral neuropathyinduced by various causes.

That is, the present invention provides an extract of the familyDioscoreaceae for preventing or treating the peripheral neuropathy.

The present invention also provides a pharmaceutical composition or foodcomposition comprising the extract or a compound isolated from theextract as an active ingredient.

Technical Solution

According to an aspect of the present invention, there is provided anextract of a family Dioscoreaceae for preventing or treating theperipheral neuropathy, the family Dioscoreaceae being at least oneselected from the group consisting of Dioscorea nipponica, Dioscoreaseptembloba, Dioscorea quinqueoloba, Dioscorea batatas, Dioscoreajaponica, Dioscorea bulbifera, Dioscorea tokoro, and Dioscorea tenuipes.

According to another aspect of the present invention, there is provideda pharmaceutical composition for preventing or treating the peripheralneuropathy, which comprises a therapeutically effective amount of theextract; and a pharmaceutically acceptable carrier.

According to still another aspect of the present invention, there isprovided a food composition for preventing or treating the peripheralneuropathy, which comprises the extract as an active ingredient.

According to still another aspect of the present invention, there isprovided a pharmaceutical composition for preventing or treating theperipheral neuropathy, which comprises a therapeutically effectiveamount of a compound represented by Formula 1 or salt thereof; and apharmaceutically acceptable carrier:

wherein R is a hydrogen atom, a C₁-C₄ alkyl group, or a saccharide.

According to still another aspect of the present invention, there isprovided a food composition for preventing or treating the peripheralneuropathy, which comprises the compound represented by Formula 1 orsalt thereof as an active ingredient.

Advantageous Effects

An extract of the family Dioscoreaceae and/or a compound isolated fromthe extract induces an endogenous nerve growth factor in an organism sothat it can be used in a wide range of applications for preventing ortreating peripheral neuropathy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 and FIG. 2 shows the effect of the compound isolated from anextract according to an embodiment of the present invention on neuriteoutgrowth.

FIG. 3 is a graph illustrating a change in the level of a nerve growthfactor (NGF) in normal mouse serum resulting from administration of acompound isolated from an extract according to an embodiment of thepresent invention.

FIG. 4 is a graph illustrating a change in the level of a NGF in theserum of a diabetes-induced mouse resulting from administration of anextract according to an embodiment of the present invention and acompound isolated from the extract.

FIG. 5 and FIG. 6 illustrates the effect of a compound isolated from anextract according to an embodiment of the present invention on thetransmission speed of the motor nerve and sensory nerve in a sciaticnerve of diabetes-induced mice (FIG. 5: DG treatment for one-month, FIG.6: DG treatment for two-month)

FIG. 7 is a graph illustrating a change in the level of a NGF in thesciatic nerve of a diabetes-induced mouse resulting from administrationof a compound isolated from an extract according to an embodiment of thepresent invention.

FIG. 8 is a graph illustrating a change in the level of sorbitol in thesciatic nerve of a diabetes-induced mouse resulting from administrationof a compound isolated from an extract according to an embodiment of thepresent invention.

FIG. 9 is images illustrating histological changes in the sciatic nerveof a diabetes-induced mouse resulting from administration of an extractaccording to an embodiment of the present invention and a compoundisolated from the extract.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present specification, the term “peripheral neuropathy” refers toa condition of peripheral nerves (motor nerves, sensory nerves, andautonomic verves) injured by various causes. The peripheral neuropathymay be subdivided into the mono-neuropathy and the poly-neuropathy (alsocalled as multiple neuropathy). The multiple neuropathy includes anyneuropathy caused by, metabolic diseases (e.g., diabetes, renal failure,hypothyroidism), drugs (e.g., antitumor agents, antituberculosis drugs)or toxic substance intoxication (e.g., Pb, organic solvents),malnutrition (e.g., vitamin deficiency, alcoholism), connective tissuedisorders (e.g., rheumatoid arthritis, systemic lupus erythematosus),inflammatory diseases (Guillain-Barre syndrome), or geneticallydetermined neuropathy. In addition, the multiple neuropathy may includea neuropathy caused by genetic factors and cancers.

An extract of the family Dioscoreaceae according to an embodiment of thepresent invention or a compound isolated from the extract derivesneurite outgrowth and increases the amount of an endogenous nerve growthfactor secreted, so that nerves of the peripheral nervous system can beeffectively differentiated, protected, and reinnervated. In particular,the extract and/or the compound allow oral administration, which improvepatients medication compliance.

The present invention provides an extract of the family Dioscoreaceaefor preventing or treating the peripheral neuropathy.

The family Dioscoreaceae is at least one selected from Dioscoreanipponica, Dioscorea septembloba, Dioscorea quinqueoloba, Dioscoreabatatas, Dioscorea japonica, Dioscorea bulbifera, Dioscorea tokoro, andDioscorea tenuipes. Preferably, the family Dioscoreaceae is Dioscoreanipponica, Dioscorea quinqueoloba, and/or Dioscorea tokoro. Morepreferably, the family Dioscoreaceae is Dioscorea nipponica.

The extract according to the present invention can be obtained throughan extraction process which includes extracting a whole part, root, oraerial part (for example, leave or stem) of the family Dioscoreaceaewith an extraction solvent (first extraction solvent) selected from thegroup consisting of water, C₁-C₄ alcohol, and a mixture of water and aC₁-C₄ alcohol. For example, the extract of the family Dioscoreaceae canbe obtained by extracting the root of the family Dioscoreaceae with thefirst extraction solvent. The first extraction solvent may be a mixturesolvent of water and methanol or a mixture solvent of water and ethanol.

In the extraction process, the whole part, root, or aerial part,preferably, the root of the family Dioscoreaceae is cut into smallsections, and then extracted with the first extraction solvent. At thistime, the amount of the first extraction solvent may be 1 to 20 times,preferably about 3 to 10 times, greater than that of the familyDioscoreaceae.

The first extract solvent may be a mixture solvent of water and methanol(for example, about 85% methanol solution) or a mixture solvent of waterand ethanol (for example, about 85% ethanol solution). The extraction isnot affected by temperature, and can be performed at various temperatureranges, such as a temperature of 15° C. to 100° C. The extraction can beperformed by cold extraction, hot extraction, superfluid extraction,centrifugal extraction, ultrasonic extraction, or reflux coolingextraction. The extraction time may vary according to the extractionmethod. For example, the extraction can be performed once or multipletimes for about 1 hour to 10 days. Preferably, the extraction can beperformed twice or three times at room temperature for about 2 daysusing the first extraction. The extract obtained by extraction with thefirst extraction solvent can be a liquid form in which impurities in theextract are removed using a conventional method, e.g., filtration, or apowder form obtained by concentrating under reduced pressure or dryingthe liquid extract using a conventional method.

In addition, when needed, the extraction process may further includeobtaining a fraction having higher contents of active ingredients. Thatis, the extraction process further includes: dispersing the extractobtained by the extraction with the first extraction solvent in water;and extracting the resultant solution with water saturated C₁-C₄ alcohol(second extraction solvent), thereby increasing the contents of activeingredients in the obtained extract.

When the extract obtained by extracting with the first extractionsolvent is dispersed in water, a liquid form per se obtained by theextraction with the first extraction solvent may be dispersed in water,or a powder form obtained by concentrating the liquid extract underreduced pressure and/or drying the liquid extract using a conventionalmethod may be dispersed in water.

The water saturated C₁-C₄ alcohol (second extraction solvent) may bewater saturated butanol.

The present invention includes, within its scope, a compositioncomprising a compound isolated from the extract, i.e., a steroidalsaponin or steroidal sapogenin. That is, the present invention includesa pharmaceutical composition for preventing or treating the peripheralneuropathy, which comprises a therapeutically effective amount of acompound represented by Formula 1 or salt thereof; and apharmaceutically acceptable carrier:

wherein R is a hydrogen atom, a C₁-C₄ alkyl group, or a saccharide.

In the compound of Formula 1, R may be hydrogen or methyl, preferablyhydrogen. That is, the compound of Formula 1 can be3-β,25R-spirost-5-en-3-ol.

The saccharide can be monosaccharide, disaccharide, or polysaccharide,such as glucose, fructose, mannose, galactose, ribose, cellulose,glycogen, sucrose, maltose, and lactose.

The salt of the compound of Formula 1 can be a conventional inorganicacid and/or organic acid addition salt prepared from steroidal saponinor sapogenin compounds. Examples of the salt of the compound of Formula1 include salts disclosed in International Laid-open Patent PublicationNo. WO2003/082893. These salts can be prepared in situ during finalseparating and purifying processes of a compound. In particular, an acidaddition salt can be prepared by reacting a refined compound in a freebase form with a suitable organic or inorganic acid and then separatingthe produced salt (see S. M. Berge, et al., Pharmaceutical Salts, J.Pharm. Sci., 66: p. 1-19(1977)). International Laid-open PatentPublication No. WO2003/082893 and the journal of. M. Berge, et al. areused as a reference in the present invention. A base addition salt canbe prepared by reacting a refined compound in an acid form with asuitable organic or inorganic base and separating the produced salt. Thebase addition salt can be a pharmaceutically acceptable metal or aminesalt. The acid addition salt can be a salt prepared from an acidselected from hydrochloric acid, sulfuric acid, phosphoric acid, andnitric acid. The base addition salt can be a salt prepared from a baseselected from sodium hydroxide, potassium hydroxide, and ammoniumhydroxide.

The compound of Formula 1 can be isolated from the extract according toan embodiment of the present invention, synthesized using a known method(see Herbert O. House, Modern Synthetic Reactions, The Benjamin CummingsPublishing Company, 1972), or commercially obtained (Sigma Co., USA.).

The method for isolating the compound of Formula 1 from the extract mayinclude an acid hydrolysis process and an recrystallization processusing the extract obtained according to an embodiment of the presentinvention (for example, an extract obtained using the first extractionsolvent and the second extraction solvent.) For example, the method forisolating the compound of Formula 1 from the extract may include:hydrolyzing with an acid, such as 2.5N hydrochloric acid, at atemperature of 50 to 150° C., preferably at about 94° C., for 30 minutesto 3 days, preferably for about 4 hours; solvent extracting the obtainedhydrolysate (that is, aglycone sapogenin) with an organic solvent, suchas chloroform, acetone, benzene, or xylene, for 1 to 60 minutes,preferably for about 15 minutes, and then separating the organic layer;when needed, concentrating the separated organic layer at a temperatureof 10 to 100° C., preferably 30 to 35° C.; recrystallizing the organiclayer or the concentrated solution of the organic layer with C₁-C₄alcohol or C₁-C₄ alcoholic solution (for example, 95% ethanol solution);and, washing the obtained precipitate with water when needed, andrecrystallizing with acetone.

The present invention provides a pharmaceutical composition forpreventing or treating the peripheral neuropathy, which comprises atherapeutically effective amount of an extract of the familyDioscoreaceae or a compound of Formula 1 or salt thereof; or apharmaceutically acceptable carrier.

The pharmaceutical composition according to the present inventionincludes a pharmaceutically acceptable carrier, and can be formulatedinto oral dosage form, external dosage form, suppository, and sterileinjection solution, such as powders, granules, tablets, capsules,suspensions, emulsions, syrups, or aerosols. The pharmaceuticallyacceptable carrier can be lactose, dextrose, sucrose, sorbitol,mannitol, xylitol, erythritol, maltitol, starch, acacia rubber,alginate, gelatin, calcium phosphate, calcium silicate, cellulose,methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone,water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesiumstearate, or mineral oil. The pharmaceutical composition may furtherinclude a diluent or an excipient, such as filler, expander, binder,humectant, disintegrant, or surfactant. A solid oral formulation can bea tablet, a pill, a powder, a granule, or a capsule. Such solidformulations may include at least one excipient selected from, forexample, starch, calcium carbonate, sucrose, lactose, and gelatin. Inaddition, such solid formulations may further include a lubricant, suchas magnesium stearate or talc. A liquid oral formulation can be asuspension, a solution, an emulsion, or syrup. In addition, the liquidoral formulation may include a diluent, such as water, liquid paraffine;humectant; sweetening agent; odorant; or preservative. A parenteralformulation can be a sterile aqueous solution, a non-aqueous solution, asuspension, an emulsion, a lyophilized formulation, or a suppository.Non-aqueous solvents or suspending agents can be propylene glycol,polyethylene glycol, natural oil, such as olive oil, or injectableesters, such as ethylolate. Vehicles for suppository can be witepsol,macrogol, Tween 61, cacao butter, Laurin, or glycerogelatine.

In the pharmaceutical composition according to the present invention, adose of the extract of the family Dioscoreaceae or the compound ofFormula 1 may vary depending on patient's state or body weight,seriousness of disease, dosage forms, administration routes, and theperiod of administration, and can be appropriately determined by aperson having ordinary skill in the art. For example, the extract of thefamily Dioscoreaceae or the compound of Formula 1 can be administered inan amount of 0.0001 to 1000 mg/kg, preferably 0.001 to 1000 mg/kg, perday. The administration can be completed once or through several timesper day. In the pharmaceutical composition according to the presentinvention, the amount of the extract of the family Dioscoreaceae or thecompound of Formula 1 may be in the range of 0.001 to 50% by weightbased on 100% by weight of the pharmaceutical composition.

The pharmaceutical composition can be administered to mammals, such asrats, mouse, livestock, or human beings, through various routs, e.g.,orally, rectally, intravenously, intramuscularly, subcutaneously,through intrauterine dura mater injection, or throughintracerebroventricular injection.

The present invention includes, within its scope, a food composition forpreventing or treating the peripheral neuropathy, which comprises anextract of the family Dioscoreaceae or the compound of Formula 1 as anactive ingredient.

The food composition according to the present invention can be used as ahealth functional food. According to Article 6727 of Korean HealthFunctional Food law, the “health functional food” refers to a food whichis produced and processed using a source or component that carries outgood functions on the human body. The “function” refers to an intakepurporting to attain good health effects, that is, a nutrient controlwith respect to the structure and function of the human body or aphysiological operation.

The food composition according to the present invention can include aconventional food additive. The conformity of the “food additive” isdetermined, as long as there are no other regulations, in considerationwith the standard and criteria of the corresponding item according tothe general rule of the food additives codex and general tests approvedby Korea Food & Drug Administration.

The items listed on the “food additives codex” include a chemicallysynthesized substance, such as ketone, glycine, potassium citrate,nicotinic acid, or cinnamic acid; natural additives, such as persimmoncolor, an extract of licorice, crystalline cellulose, caoliang color, orguar gum; or mixed formulation, such as L-sodium glutamate formulation,alkali additives for noodles, preservatives, or tar color formulation.

The food composition according to the present invention may include theextract of the family Dioscoreaceae or the compound of Formula 1 in anamount of 0.01 to 95% by weight, preferably 1 to 80% by weight, based on100% by weight of the food composition, in order to prevent and/or treatthe peripheral neuropathy. In addition, in order to prevent and/or treatthe peripheral neuropathy, the food composition can be produced andprocessed into tablets, capsules, powder, granule, liquid phase, orpills.

For example, in order to produce a health functional food in a tabletform, a mixture of the extract of the family Dioscoreaceae or thecompound of Formula 1, an excipient, a binder, a disintegrant, and otheradditives can be granulated using a conventional method, and thencompression molding process is preformed with a lubricant.Alternatively, the mixture can be directly subjected to the compressionmolding process. In addition, when needed, the health formulated food ina tablet form may include sweetening agents, and when needed, the healthformulated food in a tablet form can be coated with coating materials.

Among health functional foods in a capsule form, a hard capsuleformulation can be produced by filling a conventional hard capsule witha mixture of the extract of family Dioscoreaceae or the compound ofFormula 1 and an additive, such as an excipient, or granules of themixture, or coated granules of the mixture; and a soft capsuleformulation can be produced by filling a capsule support of gelatin witha mixture of the extract of family Dioscoreaceae or the compound ofFormula 1 and an additive, such as an excipient. When needed, the softcapsule formulation can include plasticizer, such as glycerin orsorbitol, a coloring agent, and a preservative.

A health functional food in a pill form can be produced by molding amixture of the extract of family Dioscoreaceae or the compound ofFormula 1, an excipient, a binder, and a disintegrant using a suitablemethod. When needed, the health functional food in a pill form can becoated with white sugar or other coating materials, or can be coveredwith starch, talc, or other materials.

A health functional food in a granule form can be produced bygranulating a mixture of the extract of family Dioscoreaceae or thecompound of Formula 1, an excipient, a binder, and a disintegrant usinga suitable method. When needed, the health functional food in a granuleform can include a flavoring agent and a sweetening agent.

The excipient, the binder, the disintegrant, the lubricant, thesweetening agent, and the flavoring agent used in the present inventioncan be defined as corresponding materials having the same or similarfunctions disclosed in references known in the art (The Koreanpharmacopoeia review, Moonsungsa Publication Co., Korea PharmaceuticalUniversity Association, Fifth edition, p 33-48, 1989).

MODE FOR THE INVENTION

The present invention will be described in further detail with referenceto the following examples. These examples are for illustrative purposesonly and are not intended to limit the scope of the present invention.

Example 1 Preparation of Extract

Dioscorea Nipponica was dried and the root thereof was cut into smallsections. The 500 g of the sample was added to 10 l of 85% methanolsolution and then extracted three times (each for 2 hours) at roomtemperature. Such an extraction process was repeated twice. Theresultant supernatants were collected and concentrated under reducedpressure, thereby obtaining 74 g of a crude extract.

The 74 g of the crude extract was suspended in 1 l of distilled water, 1l of water-saturated butanol was added thereto, and then, the generatedorganic layer was separated, which was repeated five times. The obtainedorganic layers were collected altogether and dried under reducedpressure. As a result, 17 g of the extract of Dioscorea Nipponica wasobtained.

Example 2 Preparation of Extract

Dioscorea quinqueoloba was dried and the root thereof was cut into smallsections. The 500 g of the sample was added to 5 l of 85% ethanolsolution and then extracted three times (each for 2 hours) at roomtemperature. Such an extraction process was repeated twice. Theresultant supernatants were collected and concentrated under reducedpressure, thereby obtaining 90 g of a crude extract.

The 90 g of the crude extract was suspended in 1 l of distilled water, 1l of water-saturated butanol was added thereto, and then, the generatedorganic layer was separated, which was repeated five times. The obtainedorganic layers were collected altogether and dried under reducedpressure. As a result, 26 g of the extract of Dioscorea quinqueoloba wasobtained.

Example 3 Preparation of Extract

Dioscorea tokoro was dried and the root thereof was cut into smallsections. The 300 g of the sample was added to 3 l of 85% ethanolsolution and then extracted three times (each for 2 hours) at roomtemperature. Such an extraction process was repeated twice. Theresultant supernatants were collected and concentrated under reducedpressure, thereby obtaining 35 g of a crude extract.

The 35 g of the crude extract was suspended in 0.5 l of distilled water,0.5 l of water-saturated butanol was added thereto, and then, thegenerated organic layer was separated, which was repeated five times.The obtained organic layers were collected altogether and dried underreduced pressure. As a result, 11 g of the extract of Dioscorea tokorowas obtained.

Example 4 Separation of Active Compound

1 g of the extract of Dioscorea nipponica obtained in Example 1 washydrolyzed at 94° C. for four hours by adding 10 ml of 2.5N HCl 10 mlthereto. Then, the resultant hydrolysate was extracted with 10 ml ofchloroform for 15 minutes. The chloroform layer was separated, filtered,and then concentrated under reduced pressure at a temperature of 30-35°C. The obtained residue was recrystallized at 4° C. using 5 ml of 95%ethanol solution. The recrystallized precipitate was filtered, washedwith water, recrystallized at 4° C. using 3 ml of acetone, and thenfiltered to obtain about 100 mg of the precipitate. The precipitate wasidentified as 3beta, 25R-spirost-5-en-3-ol represented by Formula 2.

-   -   (1) Formula: C₂₇H₄₂O₃    -   (2) Molecular Weight: 414.62    -   (3) Melting Point: 204-207° C.    -   (4) [α]²⁵ _(D)−129    -   (5) NMR Data: refer to Table 1

TABLE 1 ¹³C Chemical shift (δ)^(a) ¹H Chemical shift (δ)^(a) C-137.6-37.7 H-1 1.60-1.70; 0.87-0.91 (m, o) C-2 30.3-30.4 H-2 2.00-2.03;1.75-1.79 (m, o) C-3 78.1-78.7 H-3 3.81-3.90 (m, J = 4.7-5.6 Hz) C-438.9-39.5 H-4 2.57-2.29 (m, J = 13-14 Hz; J = 5.0; 12.0 Hz) C-5140.9-141.1 C-6 121.9-122.0 H-6 5.23-5.26 (d, J = 4.6-5.4 Hz) C-732.4-32.5 H-7 1.77-1.82; 1.41-1.46 (m, o) C-8 31.8-31.9 H-8 1.44-1.48(m, o) C-9 50.4-50.5 H-9 0.80-0.83 (m) C-10 37.2-37.3 C-11 ~21.3 H-111.35-1.38 (m, o) C-12 40.0-40.1 H-12 1.60-1.62 (m, o) C-13 40.6-40.7C-14 56.8-56.8 H-14 1.01 (m, o) C-15 32.4-32.5 H-15 1.95-1.98 (m, J =5.9-6.1 Hz); 1.31-1.35 (m, o) C-16 81.3-81.3 H-16 4.45-4.48 (m, J =7.0-7.4 Hz) C-17 63.0-63.1 H-17 1.72-1.75 (m, o) C-18 16.5-16.6 H-180.73-0.76 (s) C-19 19.6-19.6 H-19 0.80-1.00 (s) C-20 42.1-42.2 H-20 1.88(m) C-21 15.2-15.2 H-21 1.05-1.07 (d, J = 6.8-7.2 Hz) C-22 109.4-109.5C-23 32.0-32.0 H-23 1.57-1.63 (m, o) C-24 29.4-29.5 H-24 1.48-1.52 (m,o) C-25 30.8-30.8 H-25 1.50 (m, o) C-26 67.0-67.1 H-26 3.50-3.40 (m, J =10.5; 3.0; 10.5 Hz) C-27 17.5-17.5 H-27 0.60-0.63 (d, J = 4.7-5.8 Hz)

Experimental Example 1 Measurement of Neurite Outgrowth

In an incubator with conditions including 5% CO₂ and a temperature of37° C., PC 12 cell (pheochromocytoma, ATCC Number: CRL-1721) wascultured in a RPMI 1640 medium supplemented with horse serum (10%, v/v),fetal bovine serum (5%, v/v), and 1% penicillin-streptomycin

In order to find the effect of the compound of Formula 2 on the neuriteoutgrowth, the mediums supplemented with 2% horse serum, 1% fetal bovineserum, and 1% penicillin-streptomycin were added to each 6-well platecoated with poly-d-lysine and then the PC12 cells were inoculated in5×10⁴ cells each well. After 24 hours, these wells were treated with 10□/ml of ethanol, 10 □/□ of the compound of Formula 2, and 50 ng/□ of anerve growth factor (R&D system, USA), respectively. Then, after 48hours, the length of the neurite was measured using an inverted imagecontrast microscope (CK-2, Olympus, USA) (See FIG. 1 and FIG. 2)

Referring to FIGS. 1 and 2, the neurite outgrowth was not observed inthe ethanol-injected group (control), but the neurite outgrowth wasinduced both in the compound of Formula 2-treated group (DG) and in thenerve growth factor-treated group (NGF). Accordingly, it was found thatthe compound of Formula 2 induced differentiation of the PC 12 cell byinducing the neurite outgrowth.

Experimental Example 2 Measurement of Level of Nerve Growth Factor inMouse Serum

The compound of Formula 2 was dissolved in 0.2 ml of a solution ofdimethylsulfoxide:ethanol (3:1), and then orally administered once to7-week old male ICR mice (n=7) in an amount of 10 mg/kg. After 24 hours,the amount of an endogenous nerve growth factor was measured by ELISA.As a control group, an ICR mouse was orally administered once with 0.2ml of the dimethylsulfoxide:ethanol (3:1). Then, the amount of a nervegrowth factor in the control group was measured in the same manner asdescribed above.

Referring to FIG. 3, the compound of Formula 2-administered group (DG 10mg/kg P.O) showed a nerve growth factor in the serum about 2.5 timesgreater than the control group. Such results show that the compound ofFormula 2 can treat the neuropathy by suppressing the degeneration anddeath of neurons in a mouse and thus preventing a decrease in the numberof neurons.

Experimental Example 3 Measurement of Level of Nerve Growth Factor inSerum of Diabetes-Induced Mouse

An alloxan-induced diabetic mouse was prepared as an animal model havingdiabetic neuropathy, one of the multiple neuropathies. 7-week old maleICR mice underwent alimentary abstinence for 18 hours, and then alloxandissolved in a physiological saline was once injected to them byintraperitoneal injection in an amount of 160 mg/kg to induce diabetes.Mice that maintained their fasting blood sugar to 200 mg/dl or more forone week, that is, diabetes-induced mice were selected and then dividedinto a control group (n=10), an extract-administered group (n=10), and acompound-administered group (n=10). The control group was administeredwith 0.2 ml of a solution of dimethylsulfoxide:ethanol(3:1), theextract-administered group was administered with the extract obtainedaccording to Example 1 dissolved in a solution ofdimethylsulfoxide:ethanol (3:1) in an amount of 100 mg/kg, and thecompound-administered group was administered with the compound ofFormula 2 dissolved in a solution of dimethylsulfoxide:ethanol (3:1) inan amount of 10 mg/kg. The all groups were orally administered threetimes per one week, and the entire administration period was one month.The amount of an endogenous nerve growth factor in the serum wasmeasured by ELISA.

Referring to FIG. 4, the amounts of the endogenous nerve growth factorin the serum of both the extract-administered group (DN 100 mg/kg p.o)and the compound-administered group (DG 10 mg/kg p.o) were three andfour times higher than the control group to which only a vehicle wasinjected, respectively. Such results show that the extract and compoundaccording to the present invention can treat the diabetic neuropathy bysuppressing the degeneration and death of neurons in nerve-injureddiseases caused by diabetes and thus preventing a decrease in the numberof neurons.

Experimental Example 4 Measurement of Transmission Speed of Motor

Nerve and Sensory Nerve of Sciatic Nerve in Diabetes-induced Mouse Thetherapeutic effect of the compound according to the present invention onthe diabetic neuropathy, one of the multiple neuropathies, wasidentified by measuring the effect of the compound according to thepresent invention on the transmission speed of the motor nerve andsensory nerve in the sciatic nerve.

7-week old male ICR mice underwent alimentary abstinence for 18 hours,and then alloxan dissolved in a physiological saline was once injectedto them by intraperitoneal injection in an amount of 160 mg/kg to inducediabetes. Mice that maintained their fasting blood sugar to 200 mg/dl ormore for one week, that is, diabetes-induced mice were selected and thendivided into a control group (n=7), and a compound-administered group(n=7). The control group was administered with 0.2 ml of a solution ofdimethylsulfoxide:ethanol (3:1), and the compound-administered group wasadministered with the compound of Formula 2 dissolved in a solution ofdimethylsulfoxide:ethanol (3:1) in an amount of 10 mg/kg. 7-week oldmale ICR mice in which diabetes was not induced were grouped as a normalgroup (n=7), and the normal group was administered with 0.2 ml of asolution of dimethylsulfoxide:ethanol(3:1). The all groups were orallyadministered three times per one week, and the entire administrationperiod was 2 months. Administration-completed mice were sacrificed bycervical dislocation, and then the skin and muscle in the femoral regionwere quickly removed. Then, left and right sciatic nerves wererespectively separated in a length of 20 mm or more and stored in aphysiological saline while air flows through the physiological saline.The separated sciatic nerves were placed on a 20 mm round measurementplate. Then, a sensor and a stimulating probe were connected torespective neuroterminals and the electrical conductivity was measuredusing a digital storage oscilloscope to assess the nerve transmissionspeed (see FIGS. 5 and 6.)

In general, the myelin is a phospholipid membrane surrounding axons withseveral layers, and also called as myelin sheath. Like the plasticcoating of an electric wire, the myelin, through a white lipid material,prevents the electrical signals transmitted by neurons from leaking ordispersing. The myelin is regularly spaced between nodes of ranvier (aportion that forms nodes of myelin) at which the myelin is not formed,and surrounds axons. The electrical signal is transmitted along thespace, impulses are quickly transmitted along neurons, and the myelinincreases the electrical impulse speed. Accordingly, when the myelin isdestructed by nerve injury due to the neuropathy induced by diabetes,axons stops their function and the nerve transmission speed decreases.

During one month of a diabetes-induced period, the compound-administeredgroup (DM-DG) showed a transmission speed of the sensory nerve 25%higher than the diabetes-induced control group (DM) to which only thevehicle was administered (see FIG. 5). During two months of adiabetes-induced period, the compound-administered group (DM-DG) showeda transmission speed of the sensory nerve 45% higher than the controlgroup, and showed a transmission speed of the motor nerve 40% higherthan the control group (see FIG. 6). Accordingly, it was found that thecompound according to the present invention has a therapeutic effect onnerve injury due to the diabetic neuropathy by increasing thetransmission speed of the sensory nerve and motor nerve of adiabetes-induced mouse.

Experimental Example 5 Measurement of Level of Nerve Growth Factor inSciatic Nerve in Diabetes-induced Mouse

7-week old male ICR mice underwent alimentary abstinence for 18 hours,and then alloxan dissolved in a physiological saline was once injectedto them by intraperitoneal injection in an amount of 160 mg/kg to inducediabetes. Mice that maintained their fasting blood sugar to 200 mg/dl ormore for one week, that is, diabetes-induced mice were selected and thendivided into a control group (n=5), and a compound-administered group(n=5). The control group was administered with 0.2 ml of a solution ofdimethylsulfoxide:ethanol (3:1), and the compound-administered group wasadministered with the compound of Formula 2 dissolved in a solution ofdimethylsulfoxide:ethanol (3:1) in an amount of 10 mg/kg. The all groupswere orally administered three times per one week, and the entireadministration period was one month. The amount of an endogenous nervegrowth factor in the sciatic nerve was measured by ELISA (see FIG. 7)

Referring to FIG. 7, it was found that the nerve growth factor in thesciatic nerve of the compound-administered group (DM-DG 10 mg/kg P.O)was about 30% higher than that of the control group to which only thevehicle was injected. Accordingly, it is considered that the resultsshown in FIGS. 5 and 6 result from the nerve protecting function of thenerve growth factor.

Experimental Example 6 Measurement of Change in Amount of Sorbitol inSciatic Nerve of Diabetes-Induced Mouse

The therapeutic effect of the compound according to the presentinvention on the neuropathy was identified by measuring a change in theamount of sorbitol in the sciatic nerve.

7-week old male ICR mice underwent alimentary abstinence for 18 hours,and then alloxan dissolved in a physiological saline was once injectedto them by intraperitoneal injection in an amount of 160 mg/kg to inducediabetes. Mice that maintained their fasting blood sugar to 200 mg/dl ormore for one week, that is, diabetes-induced mice were selected and thendivided into a control group (n=3), and a compound-administered group(n=3). The control group was administered with 0.2 ml of a solution ofdimethylsulfoxide:ethanol (3:1), and the compound-administered group wasadministered with the compound of Formula 2 dissolved in a solution ofdimethylsulfoxide:ethanol (3:1) in an amount of 10 mg/kg. 7-week oldmale ICR mice in which diabetes was not induced were grouped as a normalgroup (n=3), and the normal group was administered with 0.2 ml of asolution of dimethylsulfoxide:ethanol (3:1). The normal group, thecontrol group, and the compound-administered group were orallyadministered three times per one week, and the entire administrationperiod was 2 weeks. Administration-completed mice were sacrificed bycervical dislocation, and then the amount of the sorbitol in the sciaticnerve was measured by HPLC (see FIG. 8).

A polyol pathway refers to a process in which glucose is converted tosorbitol by aldose reductase and the sorbitol is changed into fructoseby sorbitol dehydrogenase. In a hyperglycemic state, such as diabetes,excess glucose enters cells and sorbitol is generated and accumulated bythe polyol pathway. At this time, nerves can be injured by the osmoticoperation drawing water into cells. Accordingly, as a result ofmeasuring the effect of the compound according to the present inventionon the sorbitol accumulation in the sciatic nerve of thediabetes-induced mouse, as shown in FIG. 8, it was found that thesorbitol in the sciatic nerve of the diabetes-induced control group(DM-CON) was about 40% higher than that of the normal group, but thesorbitol in the sciatic nerve of the compound-administered group (DM-DG)was 10% lower than that of the diabetes-induced control group (DM-CON).Such results show that the compound according to the present inventioncan partially decrease aggressive factors causing nerve injury.

Experimental Example 7 Histological Comparison of Sciatic Nerve ofDiabetes-Induced Mouse

The therapeutic effect of the compound according to the presentinvention on the neuropathy was identified by measuring a histologicalchange of the sciatic nerve.

7-week old male ICR mice underwent alimentary abstinence for 18 hours,and then alloxan dissolved in a physiological saline was once injectedto them by intraperitoneal injection in an amount of 160 mg/kg to inducediabetes. Mice that maintained their fasting blood sugar to 200 mg/dl ormore for one week, that is, diabetes-induced mice were selected and thendivided into a control group (n=5), an extract-administered group (n=5),and a compound-administered group (n=3). The control group was injectedwith 0.2 ml of a solution of dimethylsulfoxide:ethanol (3:1), theextract-administered group was administered with the extract obtainedaccording to Example 1 dissolved in a solution ofdimethylsulfoxide:ethanol (3:1) in an amount of 100 mg/kg, and thecompound-administered group was administered with the compound ofFormula 2 dissolved in a solution of dimethylsulfoxide:ethanol (3:1) inan amount of 10 mg/kg. All groups were treated three times by oraladministration per one week, and the entire administration period was 2months. Administration-completed mice were sacrificed by cervicaldislocation, and then the sciatic nerve was separated, dyed according tothe following conditions, and then observed using a 600×, 1200× confocalmicroscope.

-   -   (1) prefixing: 2.5% glutaraldehyde, 12 or more hours    -   (2) washing: pH 7.4, 0.1M phosphate buffer solution, 15        minutes/twice    -   (3) postfixing: 1% OsO₄ osmium tetroxide, 60 minutes    -   (4) washing: pH 7.4, 0.1M phosphoate buffer solution, 5        minutes/twice    -   (5) dehydrating:        -   50, 70, 80, 90% alcohol: 10 minutes/each        -   100% alcohol: 15 minutes/twice    -   (6) substituting: prophylene oxide, 15 minutes/twice    -   (7) permeating: propylene oxide (1), EPOK 812 (2), 12 or more        hours    -   (8) embedding: refined EPOK 812 (80° C. polymerization): 12 or        more hours    -   (9) sectioning: semi-thin section, 35-95    -   (10) dyeing: 1% toluidine blue

FIGS. 9A and 9B show the results obtained with 600 magnification (9A)and 1200 magnification (9B), respectively. In the diabetes-induced group(DM), the axon and the myelin in the central part of the sciatic nervewere significantly destructed. However, in the compound-administeredgroup (DM-DG) and the extract-administered group (DM-DN), the axon andthe myelin in the central part of the sciatic nerve were clearlyobserved. Such results show that the extract or compound according tothe present invention can protect and treat nerves injured by theneuropathy.

The compound according to the present invention was formulated into thefollowing forms. However, these formulation examples are forillustrative purposes only and are not intended to limit the scope ofthe present invention.

Formulation Example 1 Tablet Formation

Compound of Formula 2 200

Lactose 100

Starch 100

Magnesium stearate proper These components were mixed and compressedinto tablets according to a conventional tablet formulating method.

Formulation Example 2 Liquid Formulation

Compound of Formula 2 1000

CMC-Na 20 g Isomerized sugar 20 g Lemon flavor proper

Purified water was added so that the volume of the entire solution was1000

. These components were mixed according to a conventional liquidformulation method, filled into a brown bottle, and sterilized, therebyproducing the liquid formulation.

Formulation Example 3 Capsule Formulation

Compound of Formula 2 300

Crystalline cellulose 3

Lactose 14.8

Magnesium stearate 0.2

These components were mixed according to a conventional capsuleformulating method, and then filled to a gelatin capsule, therebyproducing the capsule formulation.

Formulation Example 4 Injection Formulation

Compound of Formula 2 300

Mannitol 180

Injectable sterile distilled water 2974

Na₂HPO₄12H₂O 26

An injection containing the components having amounts described aboveper one ample (2

was manufactured according to a conventional injection manufacturingprocess.

1. An extract of a family Dioscoreaceae for preventing or treating theperipheral neuropathy, the family Dioscoreaceae being at least oneselected from the group consisting of Dioscorea nipponica, Dioscoreaseptembloba, Dioscorea quinqueoloba, Dioscorea batatas, Dioscoreajaponica, Dioscorea bulbifera, Dioscorea tokoro, and Dioscorea tenuipes.2. The extract of claim 1, wherein the family Dioscoreaceae is Dioscoreanipponica, Dioscorea quinqueoloba, or Dioscorea tokoro.
 3. The extractof claim 1, wherein the peripheral neuropathy is the multipleneuropathy.
 4. The extract of claim 1, wherein the extract is obtainedby performing an extraction process which comprises extracting a wholepart, root, or aerial part of the family Dioscoreaceae with a firstextraction solvent selected from the group consisting of water, C₁-C₄alcohol, and a mixture of water and a C₁-C₄ alcohol.
 5. The extract ofclaim 4, wherein the extract is obtained by performing an extractionprocess which comprises extracting the root of the family Dioscoreaceaewith the first extraction solvent.
 6. The extract of claim 4, whereinthe first extraction solvent is a mixture of water and methanol or amixture of water and ethanol.
 7. The extract of claim 4, wherein theextraction process further comprises dispersing the extract obtained bythe extraction with the first extraction solvent in water, andextracting with a water-saturated C₁-C₄ alcohol (second extractionsolvent).
 8. The extract of claim 7, wherein the second extractionsolvent is water-saturated butanol.
 9. A pharmaceutical composition forpreventing or treating the peripheral neuropathy, which comprises atherapeutically effective amount of the extract of claim 1, and apharmaceutically acceptable carrier.
 10. A food composition forpreventing or treating the peripheral neuropathy, which comprises theextract of claim 1, as an active ingredient.
 11. A pharmaceuticalcomposition for preventing or treating the peripheral neuropathy, whichcomprises a therapeutically effective amount of a compound representedby Formula 1 or salt thereof; and a pharmaceutically acceptable carrier:

wherein R is a hydrogen atom, a C₁-C₄ alkyl group, or a saccharide. 12.The pharmaceutical composition of claim 11, wherein R is a hydrogenatom.
 13. The pharmaceutical composition of claim 11, wherein thesaccharide is selected from the group consisting of glucose, fructose,mannose, galactose, ribose, cellulose, glycogen, sucrose, maltose, andlactose.
 14. A food composition for preventing or treating theperipheral neuropathy, which comprises a compound represented by Formula1 or salt thereof as an active ingredient:

wherein R is a hydrogen atom, a C₁-C₄ alkyl group, or a saccharide. 15.The food composition of claim 14, wherein R is a hydrogen atom.
 16. Thefood composition of claim 14, wherein the saccharide is selected fromthe group consisting of glucose, fructose, mannose, galactose, ribose,cellulose, glycogen, sucrose, maltose, and lactose.